Two Photon Microscopy has found one of its main applications in the study of deep brain regions thanks to its intrinsic optical sectioning and deep penetration capabilities. One of the main limitations of deep 3D imaging is the arise of aberrations that can be reduced if not totally corrected using Adaptive Optics (AO). In this work we present the result of using a tunable acoustic gradient lens in combination with a multi actuator deformable lens placed in the back aperture pupil of the objective lens to correct for the sample aberrations.

To understand brain functions, it is important to study functional connectivity among stereoscopically distributed neurons. Since the brain is composed of 3D neuron networks, volumetric imaging with high spatiotemporal resolution is highly desirable. Two-photon microscopy (2PM) conveniently offers 3D tissue imaging with sub-micrometer resolution based on its intrinsic optical sectioning and deep penetration capabilities. However, the main challenge lies in the volumetric imaging speed and contrast reduction in deep tissue due to aberration. In this study, we integrate a tunable acoustic gradient (TAG) lens and a plug-and-play adaptive-optics (AO) lens into 2PM. The former provides ~100kHz axial scan rate, achieving volumetric imaging rate in 1-10Hz range, while the latter enhances image contrast by nearly two-fold in deep brain regions via correcting both systematic and sample aberrations. The combination offers a practical approach toward high-speed, high-contrast optical volumetric imaging of brain tissues.